On the Dye-Protein Interaction of TMR-Biocytin – Streptavidin Complex: Molecular Simulations and Time-Resolved Fluorescence Quenching

The interaction of a fluorophore and a protein, as a type of chemical-protein interactions (CPIs), is important toward studying physical properties of a protein through spectroscopy. For example, the structural changes of a protein can often be followed with spectroscopic tools by utilizing such an interaction. Streptavidin-bound 5-(and-6)-tetramethylrhodamine (TMR) biocytin is one of the few systems with which CPI can actually be observed via the fluorescence of TMR. While it has been suggested that the fluorescence is quenched when the ligand is bound to the binding pocket of streptavidin and is recovered after biotin occupies the binding pocket to replace TMR, no clear evidence has been provided yet. In order to gain a better understanding of this CPI, here we propose a possible binding site of TMR in the streptavidin, and predict the binding affinity values of biotin and the TMR moiety of TMR-biocytin conjugate. We estimate that the biotin affinity (-20.2 ± 1.4 kcal/mol) is significantly higher than the TMR-biocytin affinity (-6.91 ± 0.72 kcal/mol and -8.06 ± 0.79 kcal/mol), which supports the earlier explanation on the fluorescence quenching and recovery of biotinylated dyes. We also discover that TMR cannot bind to the streptavidin pocket as perfectly as biotin does, but that it rather blocks the entrance portion of the binding pocket. Fluorescence quenching of TMR-biocytin by streptavidin is measured with time-resolved fluorescence to show that the average fluorescence lifetime of TMR-biocytin decreases from 3.54 ns to 2.54 ns in the presence of excess streptavidin. Based on the distance between TMR and the nearby tryptophan residues, we propose that the nearby tryptophan residues of streptavidin are the potential quenchers of the dye unit fluorescence.